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Biomedical subjects

D Wirth

Publications and source records attributed to D Wirth.

At least 19 recordsLinked to original sources
PubMed2006

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

Cell Line↗
PubMed2006

Outcome and quality of life after open surgery versus endoscopic stapler-assisted esophagodiverticulostomy for Zenker's diverticulum.

Two techniques for treatment of Zenker's diverticulum, endoscopic stapler-assisted esophagodiverticulostomy and open cricopharyngeal myotomy by transcervical approach, were compared with regard to patient satisfaction and quality of life. Between January 1994 and December 2004 a total of 47 patients with Zenker's diverticulum underwent surgery in our department. Besides the usual retrospective evaluation of details of surgery, all patients were sent a questionnaire on their actual complaints and quality of life according to the Gastrointestinal Quality of Life Index (GIQLI). Twenty patients had the endoscopic procedure (Group A), and 27 the open procedure (Group B). The preoperative symptoms were dysphagia in 96%, regurgitation of undigested food in 60%, cough in 19%, and pneumonia caused by recurrent aspiration in 9%. The length of surgery was on average 32 min (range 5-70 min) in Group A and 106 min (range 45-165 min) in Group B, and the length of hospital stay was 5.5 days (range 1-10 days) and 12.3 days (range 7-25 days), respectively. The results of the questionnaire showed that the preoperative symptoms had disappeared in up to 83%, and 91% in Group A and 100% in Group B would be willing to undergo surgery again. The mean GIQLI was 123 points in Group A and 118 points in Group B (healthy volunteers in the literature, 125 points). Both techniques showed good results in a long-term follow-up with regard to relief of symptoms and patient satisfaction. Both groups had an excellent Gastrointestinal Quality of Life Index, comparable to that of a healthy standard population.

Aged↗
PubMed2005

[In vitro evaluation of the sensitivity of Plasmodium falciparum to chloroquine using the deli-microtest in region of Dakar, Senegal].

This study was carried out between 2000 and 2001 in the peri-urban Pikine area located 15 km from Dakar, Senegal. The purpose was to evaluate the in vitro sensitivity of Plasmodium falciparum isolates to chloroquine, which was the recommended first-line drug for uncomplicated malaria treatment in Senegal. Testing was carried out using the double-site enzyme-linked lactate dehydrogenase imnunosorbent (DELI) microtest. The DELI-microtest is an ELISA method using 2 monoclonal antibodies against 2 antigenic sites of the specific P. falciparum lactate dehydrogenase (LDH) enzyme. The level of LDH is proportional to the extent of parasite growth. P. falciparum isolates were cultured in 96-well plates in RPMI 1640 medium supplemented with hypoxanthine and albumax, in the presence of chloroquine sulphate concentrations ranging from 5.6 nM/L to 2870.8 nM/L. Plates were incubated in a candle-jar for 48 hours at 37 degrees C and frozen at -20 degrees C. The DELI-microtest was performed using the supernatant of hemolysed cultures. The amount of pLDH released was evaluated based on optical density. The chloroquine sensitivity of the isolate was estimated based on IC50 with a cut-off of <100 nM/L. Geometric mean IC50 values were 41 nM/L (range: 4.8 nM/L to 1435 nM/L) and 135 nM/L (range: 8.63 nM/L to 2153 nM/L) in 2000 and 2001 respectively. This study demonstrated a dramatic increase in the in vitro resistance of P. falciparum to chloroquine from 30% in 2000 to 59.6% in 2001. These findings suggest that regular surveillance of in vitro drug resistance is important to predict in vivo drug resistance and allow timely changes in public health recommendations.

Animals↗
PubMed2003

Effect of polystyrene particles on lung microvascular permeability in isolated perfused rabbit lungs: role of size and surface properties.

The aim of this study was to investigate the role of particle number, total surface area, mass and surface chemical groups in (K(f,c)) changes. The lung effects of four different fine (110 nm) and ultrafine (24 nm) polystyrene particles have been tested in an isolated perfused rabbit lung model. Pulmonary microvascular permeability (K(f,c)) modifications were measured in response to intratracheal particle challenge. Polystyrene particles, mainly located in alveolar spaces and macrophages, induced a K(f,c) increase that was related to the total surface area and number of particles rather than to the instilled mass. Moreover, the positively charged amine-modified polystyrene particles were more effective in the K(f,c) response than the negatively charged carboxylate-modified polystyrene particles. We concluded that particle number and diameter that mathematically equally determined total surface area do not have the same importance in explaining the biological effects observed and that particle number could be an alternative to total surface area to describe the particle exposure. Furthermore, surface properties of polystyrene particles need to be considered to investigate the microvascular permeability changes measured in our model.

Animals↗
PubMed2003

From the germinal cells to the newborn animal: the transmission of genes and life through the generations.

The technology of reproduction progressed considerably during the last decade, leading to a certain availability of in vitro methods for fertilisation, oocyte maturation and embryo culture. The most spectacular manipulations are cloning and transgenesis. This review focuses on the early appearance of germinal cell precursors and the long-standing fate of gametes in mammals. The evident complexity and long-term programming of events in gametes and early embryos explain part of the difficulties encountered during the development of in vitro and in vivo methods such as multiple ovulation and embryo transfer (MOET), oestrus synchronisation, ovulation induction, superovulation, in vitro maturation and fertilisation, cryopreservation, transgenesis, nuclear transfer and cloning) and the occurrence of unexpected alterations of development, e.g. embryonic or fetal mortality, large-weight newborn syndrome and other dysregulations in imprinting or DNA transmission.

Animals↗
PubMed2002

Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein.

OBJECTIVE: One of the main open questions in chondrocyte transplantation is the fate of the implanted cells in vivo. We intended to establish prerequisites for such studies in animal models and to show the feasibility of this approach in rabbits. Isolated articular chondrocytes were retrovirally marked using green fluorescence protein (GFP) as a cell-specific marker in order to allow an in vivo follow-up of these cells. METHODS: Chondrocytes from rabbits, sheep, cattle and humans were isolated and infected with murine leukemia virus-derived retroviruses carrying the GFP gene. The influence of the host range of three packaging cell lines (PA317, PT67, PG13), start cell concentrations, number of cell passages and number of infection cycles on the efficiency of infection was investigated. Stability of GFP expression was followed by FACS analysis, confocal imaging and fluorescence microscopy. For in vivo follow-up of GFP expression we used marked allogeneic chondrocyte populations grown on scaffold material and implanted them into full-thickness defects in knee joints of rabbits. RESULTS: Retroviruses from all three packaging cell lines were able to infect rabbit and human chondrocytes, whereas only retroviruses released from PG13 cells were able to infect sheep and bovine chondrocytes efficiently. Optimization of the infection with these viruses resulted in efficiencies of 60-90% GFP-expressing chondrocytes. Populations of 100% marked chondrocytes were obtained by cell sorting. GFP expression stability of such marked chondrocyte populations was followed in monolayer culture and in 3-D culture on different scaffold materials. The expression of GFP was stable on all tested materials for at least 4 weeks. In monolayer culture GFP expression was stable for more than 8 months. In vivo, we observed stable GFP expression in the transplants during a four-week time course. CONCLUSION: Retroviral GFP gene transfer led to long-term expression in chondrocytes from rabbits, sheep, cattle and humans. Transgene expression and the number of implanted chondrocytes remain stable for at least 4 weeks in vivo. This method permits a rapid monitoring of chondrocytes and provides a basis for following the fate of these cells in vivo.

Animals↗
PubMed2001

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

3T3 Cells↗
PubMed2001

Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement.

Successful retroviral vector construction is still empirical. Test systems for vector efficiency are based on statistical comparison of numerous infectants with single proviral integrates, since their expression depends on the chromosomal surroundings. More reliable data would be obtained if different vector constructs were studied in an identical chromosomal context. Here, we demonstrate the use of a new method, in which chromosomal sites are provirally tagged in such a way that they can be targeted with other expression cassettes. The original tagging integrate is replaced in one step by the targeting element. This permits a reliable comparison of different retroviral vector configurations, eliminating the influence of neighboring chromosomal elements. We compared different retroviral vector types for coexpression of two genes: a vector containing an internal promoter and a vector with an internal ribosome entry site (IRES) element. In contrast to bicistronic retroviral vectors, dual-promoter proviruses exhibited rapid inactivation of the long terminal repeat (LTR)-driven gene expression. Targeted exchange of the dual-promoter provirus with a bicistronic retroviral cassette resulted in gain of expression stability. The reverse experiment confirmed this promoter interaction phenomenon since initial expression stability from a single-promoter bicistronic provirus was lost by targeted exchange with a dual-promoter cassette. In addition, targeting exchange of the dual-promoter provirus, replacing the LTR with an artificial (Tet) promoter restored expression stability. These observations, valid for various integration sites, prove the strong interaction between the LTR and the internal promoter. Our results have implications for retroviral vector design and suggest that retroviral coexpression of two genes is more predictable in the bicistronic configuration.

3T3 Cells↗
PubMed2001

Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells.

Animals↗
PubMed2001

Drugs for neglected diseases: a failure of the market and a public health failure?

Infectious diseases cause the suffering of hundreds of millions of people, especially in tropical and subtropical areas. Effective, affordable and easy-to-use medicines to fight these diseases are nearly absent. Although science and technology are sufficiently advanced to provide the necessary medicines, very few new drugs are being developed. However, drug discovery is not the major bottleneck. Today's R&D-based pharmaceutical industry is reluctant to invest in the development of drugs to treat the major diseases of the poor, because return on investment cannot be guaranteed. With national and international politics supporting a free market-based world order, financial opportunities rather than global health needs guide the direction of new drug development. Can we accept that the dearth of effective drugs for diseases that mainly affect the poor is simply the sad but inevitable consequence of a global market economy? Or is it a massive public health failure, and a failure to direct economic development for the benefit of society? An urgent reorientation of priorities in drug development and health policy is needed. The pharmaceutical industry must contribute to this effort, but national and international policies need to direct the global economy to address the true health needs of society. This requires political will, a strong commitment to prioritize health considerations over economic interests, and the enforcement of regulations and other mechanisms to stimulate essential drug development. New and creative strategies involving both the public and the private sector are needed to ensure that affordable medicines for today's neglected diseases are developed. Priority action areas include advocating an essential medicines R&D agenda, capacity-building in and technology transfer to developing countries, elaborating an adapted legal and regulatory framework, prioritizing funding for essential drug development and securing availability, accessibility, distribution and rational use of these drugs.

Communicable Diseases, Emerging↗
PubMed2000

New approaches towards ex vivo and in vivo gene therapy.

A number of hurdles have to be overcome for efficient and specific gene therapy approaches. Here, we report on two different strategies that should lead to an improvement of current protocols. A strategy is presented to tag unique chromosomal integration sites by means of retroviral infection, which can be reused for exchange with the gene of interest by action of site-specific recombinases. Targeting exchange is achieved in one step with 100% efficiency by a stringent positive selection, which makes further screening superfluous. With this strategy a predictable gene expression is obtained for foreign genes integrated into a predefined chromatin structure. A second approach aims at the stabilization of mouse retroviruses towards human serum which is a prerequisite for in vivo gene therapy protocols. To stabilize murine leukemia virus-based retroviruses against human serum, complement regulatory proteins were fused to the retroviral ENV proteins. This resulted in infectious and human complement-protected particles.

3T3 Cells↗
PubMed1999

Complement-protected amphotropic retroviruses from murine packaging cells.

The application of retroviruses generated from murine cells for in vivo gene therapy is restricted primarily because of the rapid inactivation of these viruses by the human complement system. To circumvent this disadvantageous property of murine retroviruses we have generated infectious amphotropic retroviruses that exhibit strong protection against human complement attack. The membrane of these viruses contains a fusion protein, DAFF2A, that is composed of the catalytic domain of the human complement regulatory protein (CRP) decay-accelerating factor (DAF) and the envelope protein of the amphotropic murine leukemia virus (MuLV) 4070A (EnvA). The fusion of two other CRPs, MCP and CD59, to the same amphotropic Env moiety did not lead to equivalent results. The fusion protein DAFF2A was stably expressed in mouse NIH 3T3-based helper cells and independently identified with either alpha-DAF MAb or alpha-Env PAb on the cell membrane. Western blot analysis confirmed the expected molecular weight of the fusion protein. Viral titers obtained from NIH 3T3 helper cell pools were 5 x 10(5) CFU for wild-type amphotropic EnvA virus and 1 x 10(5) CFU for DAFF2A virus, respectively. By blocking the catalytic domain of DAF by pretreatment with alpha-DAF MAb DAFF2A, recombinant virions could be converted to wild-type with respect to sensitivity against human serum. Since the method for producing virions that are protected against human serum should be applicable to any cell type it offers a novel tool for human in vivo gene therapy.

3T3 Cells↗
PubMed1999

G-CSF receptor mutations in patients with severe congenital neutropenia do not abrogate Jak2 activation and stat1/stat3 translocation.

Severe congenital neutropenia (SCN) is an inherited disorder of myelopoiesis, characterized by a maturation arrest at the stage of promyelocytes and myelocytes in bone marrow, and absence or low levels of mature neutrophil granulocytes in peripheral blood. Recently, studies of patients with SCN who subsequently developed acute myeloid leukemia (AML) revealed nonsense mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor (G-CSF) receptor messenger RNA. We focused our interest on the G-CSF-mediated signaling cascade to examine the consequences of the observed point mutations for the nuclear translocation of the transcription factors Stat1 and Stat3. Expression vectors encoding for truncated G-CSF receptors were transfected in the murine fibroblast cell line C243 expressing a fusion protein consisting of the transcription factor Stat1 and Stat3, respectively, and the green fluorescent protein (GFP). Nuclear translocation of the GFP fusion proteins was examined after G-CSF stimulation of the transfected cells.

Bone Marrow↗
PubMed1996

Determinants of current HIV risk behaviour among injecting drug users in Warsaw, Poland.

Of the injecting drug users (IDUs), 24% had borrowed, and 37% had passed on syringes in the previous 6 months. In logistic regression analysis, current borrowing of syringes was significantly associated with a shorter duration of injecting drug use, a higher number of drug-injecting sex partners in the previous 6 months, and with current lending of syringes. A majority of the IDUs have modified previous HIV risk behaviour but these modifications are insufficient and not sustained over time. Intensified AIDS prevention measures for IDUs in Poland are needed.

Adult↗